The native biosynthetic scheme of insulin involves an efficient folding/disulfides formation in a single chain precursor proinsulin molecule and subsequent enzymatic removal of the C-peptide to give mature insulin (Oyer et al., (1971) J. Biol. Chem. 246:1375-1386; and Kemmler et al., (1971) J. Biol. Chem. 246: 6786-6791). A proinsulin-based approach has been currently adopted in the recombinant production of insulin (Frank et al., MMW Munch. Med. Wochenschr (1983) 125(Suppl 1):14-20; and Thim et al. (1986) Proc. Natl. Acad. Sci. USA 83:6766-6770).
In current recombinant synthesis of human insulin (and analogs) as drug manufactures, the chemical diversification is limited to natural amino acids (Mayer et al., (2007) Biopolymers (Peptide Science) 88:687-713): Conversely, total chemical synthesis of human insulin permits the incorporation essentially any non-natural structure into the molecule, an apparent advantage that allows for full exploration of the medicinal chemistry of this important therapeutic molecule. However, an efficient chemical synthesis approach to human insulin is lacking (Mayer et al., (2007) supra). This apparent deficiency hampers not only the cost-effective chemical manufacture of insulin, but also the development of next generation insulin analogs containing non-natural chemical structure.
The difficulty of chemical insulin synthesis is mainly attributed to a low yield recombination of A- and B-chains via interchain disulfides formation. In addition, the disulfides-forming reaction requires an excess amount of A-chain (Hua et al. (2002) J. Biol. Chem. 277:43443-43453). An initial set of total chemical syntheses of insulin involved the inefficiency in three disulfides formation from separated A- and B-chains which were prepared by segment condensation (Meienhofer at al. (1963) Z. Naturforsch. 18b:1120-1121; Katsoyannis et al., (1964) J. Am. Chem. Soc. 86:930-932; and Du et al., (1965) Sci. Sin. 14:229-236) or solid phase peptide synthesis (Marglin et al., (1966) J. Am. Chem. Soc. 88:5051-5052). An alternative methodology with chemically directed formation of the disulfide bonds (Sieber et al. (1977) Helv. Chim. Acta 60:27-37; and Akaji et al., (1993) J. Am. Chem. Soc. 115:11384-11392) is complicated and has not found widespread use.
An alternative is to use a chemical tether to mimic the effect of covalently linking the A- and B-chains as found in proinsulin. Most of the previously studied chemically tethered insulin precursors have involved covalent linking of the N-terminal of the insulin A chain to the side chain of LysB29, near the C-terminal of the insulin B-chain (Brandenburg, D. & Wollmer, A. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354:613-627; Brandenburg et al. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354:1521-1524; Geiger, R. & Obermeier, R. (1973) Biochem. Biophys. Res. Commun. 55:60-66; Obermeier, R. & Obermeier, R. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356:1631-1634; and Busse, W-D. & Carpenter, F. H. (1976) Biochemistry 15:1649-1657). With a view to an efficient total synthesis of human insulin and analogues, a variety of different length chemical tethers between these two functionalities have been explored with both non-cleavable (Brandenburg, D. & Wollmer; A. (1973) supra) and cleavable tethers (Brandenburg et al. (1973) supra; Geiger et al. (1973) supra; Obermeier et al. (1975) supra; and Busse, W-D. & Carpenter, F. H. (1976) supra). It was found that tethers as short as 8 carbon atoms were effective in promoting high yield folding/disulfide formation (Brandenburg, D. & Wollmer, A. (1973) supra).
Recently, there have been attempts to extend the chemical tether approach to a more effective total chemical synthesis of insulin (Tofteng et al. (2008) Chem Bio Chem 9:2989-2996; and Sohma, Y. & Kent, S. B. H., “Biomimetic synthesis of lispro insulin via a chemically synthesized ‘mini-proinsulin’ prepared by oxime-forming ligation” Submitted). For example, synthesis of human insulin via chemically synthesized ‘mini-proinsulin’ prepared by oxime-forming ligation has been reported, in which an introduced temporary ‘chemical tether’ that links the N-terminal of the A chain to the C-terminal of the B chain, permitted the folding/formation of disulfides with high efficiency (Sohma, Y. & Kent, S. B. H, Submitted, supra). However, this later approach involved a relatively long/complicated chemical tether which made the synthesis laborious. In addition, it was necessary to remove the chemical tether enzymatically in a later step. Thus, various chemical production strategies reported to date for insulin are still far from practical for the efficient generation of insulin chemical analogues.
Ideal features of an optimal chemically tethered mini-proinsulin would include: straightforward preparation by existing synthetic methods; efficient folding/disulfide formation; and, ready chemical conversion to mature insulin. The present invention addresses these and other needs.